MAP Kinase (ERK1/2) Activity Assay Kit

Transcription

1 MAP Kinase (ERK/2) Activity Assay Kit For 96 tests Cat. No. SGT45 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA & Canada Phone: +(800) Fax: + (909) Europe +44 (0) Australia Germany ISO Registered Worldwide custserv@chemicon.com techserv@chemicon.com

2 Introduction Upon growth factor or cytokine treatment, transmission of stimulatory signals from receptors to the nuclear targets appears to involve the regulation of the activity of a family of kinases known as MAPKs (mitogen-activated protein kinases) or ERKs (extracellular signal regulated kinases) (Figure )[,2]. For example, the ability of growth factors to promote proliferation depends on the activation of receptor tyrosine kinases, which recruit the Ras family small G proteins and lead to the sequential activation of Raf (MAPK kinase kinase), MEK (MAPK kinase), and MAPK/ERK [-4]. MAPK/ERK activity requires phosphorylation on both threonine (T85) and tyrosine (Y87) [3]. The MAP kinase cascade is an evolutionarily conserved signaling pathway that plays a central role in integrating the signals from a diverse group of extracellular stimuli and proto-oncogenes to the nucleus where activation of specific transcription factors elicits cellular responses including proliferation, differentiation, survival, and apoptosis in all eukaryotes [5, 6]. Figure

3 CHEMICON s non-radioactive MAP Kinase (ERK/2) Activity Assay provides a simple, convenient, and specific method for measuring ERK/2 activity. Testing of purified ERK enzyme, in vitro inhibitor screening and the study of ERK regulation can be performed with this assay. The assay has been validated for use with immunoprecipitated ERK-antibody complexes. Test Principle CHEMICON s non-radioactive MAP Kinase (ERK/2) Activity Assay Kit consists of a Biotinylated MBP Substrate, a purified Phospho-MBP specific monoclonal antibody, a secondary antibody conjugated to horseradish peroxidase (HRP) and other components required to perform 96 ELISA-based assays (Figure 2). The Biotinylated MBP Substrate contains multiple phosphorylation sites and can be phosphorylated by a wide range of kinases, for example, phosphorylation on Thr 97 residue by MAP kinases (ERKs). After quenching the enzyme reaction with an inhibitor, both the phosphorylated and dephosphorylated substrates are immobilized by binding to the streptavidin-coated plate. The fraction of phosphorylated substrate is visualized using a phospho-mbp monoclonal antibody and a HRP conjugated secondary antibody and an ensuing chromogenic substrate reaction. Reagents and materials supplied in the MAP Kinase Activity Assay Kit are sufficient for 96 reactions. Figure 2: Test Principle The CHEMICON MAP Kinase (ERK/2) Activity Assay Kit is intended for research use only; not for diagnostic or therapeutic applications. 2

4 Kit Components. Streptavidin-Coated Plate (Part No. 9090): One 96-well plate with 2 strips. 2. MAP Kinase Substrate (Part No. 9023): One vial contains 50 µg of biotinylated MBP at 0.5 mg/ml. 3. Mouse Anti Phospho-MBP Antibody (Part No. 9022): One 0 vial. 4. MAP Kinase Assay Buffer (Part No. 902): One 2 ml bottle (5X) containing 250 mm Tris, ph 7.5, 50 mm MgCl 2, 0.% BSA. 5. ATP/MgCl 2 Solution (Part No. 9094): One.5 ml vial (5X) containing 5 mm ATP, 50 mm MgCl 2 in TBS (25 mm Tris, 0.5 M NaCl) ph 7.2, and 0.0% thimerosal added as preservative. 6. HRP-conjugated Secondary Antibody (Part No. 902): One lyophilized vial. 7. Blocking Buffer (Part No. 9095): One 50 ml bottle (Ready to Use). 8. Wash Buffer (Part No ): One 0 ml bottle (X). 9. TMB Substrate Solution (Part No: 90028): One 2 ml bottle (Ready to Use).. Stop Solution (Part No: 6093): One 2 ml bottle (Ready to Use). Materials Not Supplied. MAP Kinase Inhibitor: Kinase inhibitors are required for stopping the kinase reaction after incubation with the MBP substrate. Several kinase inhibitors are available, for example EDTA. 2. Lysis Buffer: Such as RIPA buffer: 50 mm Tris, ph 8.0, 50 mm NaCl, 0.5 mm EDTA, mm DTT, % NP-40, 0.5% Sodium Deoxycholate, 0.% SDS, 0 µg/ml PMSF*, µg/ml Aprotinin*, 2 µg/ml Leupeptin*, 0 µm Sodium Vanadate. Note: Protease Inhibitors (PMSF*, Aprotinin*, Leupeptin*) should be added freshly from stock solution. 3. Microcentrifuge and tubes C Water Bath C Incubator well Microplate Reader 3

5 Preparation of Reagents. MAP Kinase Substrate: Immediately before use dilute the MBP stock :5 with X Kinase Assay Buffer. Do not store diluted MBP solutions. 2. 5X MAP Kinase Assay Buffer: Immediately before use add DTT to the 5X Kinase Assay Buffer (final concentration of -5 mm). 3. Mouse Anti Phospho-MBP Antibody: Immediately before use dilute the antibody :0 with Blocking Buffer. Do not store diluted solutions. 4. HRP-conjugated Secondary Antibody: Reconstitute the secondary antibody with 0 of 50% Glycerol. Aliquot and store the reconstituted antibody solution at 20 C. Immediately before use dilute the HRP conjugated secondary antibody solution :5000 with X PBS. Do not store diluted solutions. 5. Wash Buffer (X): Dilute wash buffer : with distilled water for a X Wash Buffer. Sample Preparation Various extraction and purification methods can be utilized to isolate MAP kinases from blood, solid tissue or cell culture. The following protocols are provided as EXAMPLES of suitable methods. It is strongly advised that an initial experiment should be performed to determine the proper dilution of the crude sample or purified MAP kinase to be used in subsequent experiments. Solid Tissue Extracts:. Homogenize fresh tissue (brain, liver, etc.) one to four volumes of a prechilled detergent lysis buffer (RIPA buffer). 2. Centrifuge the homogenate at 2,000 x g for minutes at 4 C to pellet the insoluble fraction. 3. Remove clear supernatant carefully from pellet. This supernatant is your total protein extract. It should be stored at 70 C. Note: Every freeze-thaw cycle will decrease the enzymatic activity of protein extract. 4

6 Cell Culture Lysates:. Remove cell media and wash once with pre-chilled PBS. 2. Adherent cells: Add ml/0 mm dish of the pre-chilled detergent lysis buffer. Place culture dish on ice for minutes. Harvest cells with rubber policeman and collect cell lysate by centrifugation at 2,000 x g for minutes. Suspension cells: Place cells on ice. Add ml/ 7 cells of pre-chilled detergent lysis buffer and lyse resuspended cells using either a homogenizer, sonication, or three cycles of freezing and thawing. 3. Transfer extracts to microcentrifuge tubes and centrifuge at 2,000 x g for minutes. 4. Collect clear lysate and store at 70 C. This supernatant is your cytosolic fraction. Immunoprecipitation:. Add -20 of anti-map Kinase antibody to 0.5- ml of cell lysate. Incubate -2 hours at 4 C on a shaking or rocking platform. 2. Add 50-0 of protein A and/or G agarose beads (50% slurry) and incubate for -4 hours at 4 C on a rocking/shaking platform. 3. Collect protein A/G - antigen - antibody complexes by centrifugation at 2000 x g for minute at 4 C. 4. Remove supernatant carefully and add ml cell lysis buffer to the beads, resuspend and incubate for minutes at 4 C on a rocking/shaking platform. 5. Collect protein A/G antigen - antibody complexes by centrifugation at 2000 x g for minute at 4 C. 6. Repeat steps 4 and 5 once more. 7. Repeat steps 4 and 5 once more, using the appropriate kinase assay buffer instead of cell lysis buffer. 8. Remove supernatant carefully. The precipitated protein can be used directly for the kinase assay (see Assay Protocol). 5

7 Assay Protocol Table : Composition of reaction mix Sample Diluted Kinase Substrate ERK Sample 5X Assay Buffer DI Water Inhibitor Protein A beads ATP/Mg Solution Total Volume Negative Control Kinase Activity (ERK in solution) Kinase Activity (Immunoprecipitate d ERK) Kinase Inhibitor 2 _ part* part* part* Add up to 50uL Add up to 50uL Add up to 50uL Add up to 50uL _ part* part* _ *( part) is stated because the exact microliter volume added will depend upon the activity of the enzyme or inhibitor or the amount of material used in IP, and this amount should be optimized for the best signals.. Prepare the assay mixture according to the table above and start the reaction by adding of 5X ATP/MgCl 2 solution. 2. Following the composition of the reaction mix is an incubation period at 30 C. The phosphorylation reaction time will vary from a few minutes to 60 minutes (determined by end user), a recommended starting reaction time is 30 minutes. 3. Terminate enzyme reaction by adding of kinase inhibitor, such as 20 mm EDTA. 4. Transfer 50 of reaction mix to Streptavidin-coated strip wells and incubate at 37 C for 30 minutes. 6

8 5. Wash four times with X Wash Buffer. A thorough washing of the plate is important to reduce background. We recommend using a multi-channel pipette to fill each well with 250 of X Wash Buffer. Fluid removal from the wells is best accomplished by inverting the plate over a sink and flicking the fluid out of the wells and then blotting the plate on clean paper towels. 6. Add 200 of Blocking Buffer Solution to each well and incubate at 37 C for 30 minutes. 7. Discard Blocking Buffer Solution and add 0 of diluted mouse anti Phospho-MBP solution to each reaction well. Incubate for hour at room temperature on a shaking platform. 8. Wash four times with X Wash Buffer according to step 5 above. 9. Discard primary antibody and add 0 of the diluted HRP conjugated secondary antibody solution to each reaction well. Incubate for 30 minutes at room temperature.. Wash four times with X Wash Buffer according to step 5 above.. Warm TMB substrate to room temperature. Add 0 of TMB Substrate to each well. Incubate at room temperature for 5 5 minutes (time determined by end user). 2. Stop the enzyme reaction by adding 0 of Stop Solution into each well, including the control wells. Results should be read immediately (color will fade over time). 3. Read absorbance of each microwell on a standard microplate reader using 450 nm as the primary wave length. Note: Background reading of Negative Control should be subtracted from the readings of the kinase reaction samples before calculating the MAP kinase activity. Storage Store kit materials at 2-8 C for up to their expiration date. 7

9 Calculation of Results Relative optical density (OD) values obtained with the CHEMICON MAP Kinase (ERK/2) Activity Assay Kit may be compared with known standards or other test samples to obtain relative activities. Therefore if required, a purified ERK or ERK2 can be used for the protein kinase activity curve. Figure 3 illustrates typical results upon using purified ERK2 kinase. Kinase reaction time is 30 minutes and incubation time of TMB substrate is 5 minutes. It is always recommended to run a negative control sample. One should use the data below for reference only. This data should not be used to interpret actual assay results. Figure 3: Example of MAP Kinase Activity Assay Purified active ERK2 was incubated with the biotinylated MBP substrate for 30 minutes at 30 C and activity was detected as described in the Assay Protocol. Unit definition: One unit is the amount of ERK2 kinase that will incorporate nmol phosphate onto the MBP substrate per minute at 30 C. OD 450nm Purified ERK2 Kinase Activity Assay ERK2 Kinase (Units) 8

10 Figure 4: ERK2 kinase activity in Cos-7 lysate COS-7 cells were transiently transfected with wild type ERK2 along with a dominant active MEK. Cells were lysed in RIPA buffer and cell lysates were prepared. The phosphorylation level of ERK2 in cell lysate was determined by a phospho-erk antibody western blot (A). ERK2 in the cell lysate was also immunoprecipitated with an anti-erk2 antibody. Activity of ERK2 kinase was determined as described in the Assay Protocol (B). A B Activity of ERK2 Immunoprecipitated from COS-7 Lysate 2.5 OD 450nm ERK2 ERK2+ MEK 9

11 References. Cano E and Mahadevan LC. (995) Trends Biochem Sci. 20, Lewis TS, Shapiro PS, and Ahn NG. (998) Advances in Cancer Research. 74, Leevers SJ and Marshall CJ. (992) EMBO., Sozeri O et al. (992) Oncogene. 7, Blenis, J. (993) Proc. Natl. Acad. Sci. U.S.A. 90, Cobb, MH et al. (994) Seminars in Cancer Biol. 5, Warranty These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. 2003: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing.

12 Cat No. SGT45 July 2003 Revision F: 437